C--H functionalisation in organic synthesis themed issue
Transcript of C--H functionalisation in organic synthesis themed issue
This article was published as part of the
C---H functionalisation in organic synthesis themed issue
Guest editors Huw M. L. Davies, Justin Du Bois and Jin-Quan Yu
Please take a look at the issue 4 2011 table of contents to
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This journal is c The Royal Society of Chemistry 2011 Chem. Soc. Rev., 2011, 40, 2003–2021 2003
Enzymatic functionalization of carbon–hydrogen bondsw
Jared C. Lewis,aPedro S. Coelho
band Frances H. Arnold*
b
Received 6th August 2010
DOI: 10.1039/c0cs00067a
The development of new catalytic methods to functionalize carbon–hydrogen (C–H) bonds
continues to progress at a rapid pace due to the significant economic and environmental benefits
of these transformations over traditional synthetic methods. In nature, enzymes catalyze regio- and
stereoselective C–H bond functionalization using transformations ranging from hydroxylation to
hydroalkylation under ambient reaction conditions. The efficiency of these enzymes relative to
analogous chemical processes has led to their increased use as biocatalysts in preparative and
industrial applications. Furthermore, unlike small molecule catalysts, enzymes can be systematically
optimized via directed evolution for a particular application and can be expressed in vivo to
augment the biosynthetic capability of living organisms. While a variety of technical challenges
must still be overcome for practical application of many enzymes for C–H bond functionalization,
continued research on natural enzymes and on novel artificial metalloenzymes will lead to improved
synthetic processes for efficient synthesis of complex molecules. In this critical review, we discuss the
most prevalent mechanistic strategies used by enzymes to functionalize non-acidic C–H bonds, the
application and evolution of these enzymes for chemical synthesis, and a number of potential
biosynthetic capabilities uniquely enabled by these powerful catalysts (110 references).
I. Introduction
Although the field of chemical synthesis has matured to a
discipline capable of generating compounds of amazing
complexity, inefficiencies in the synthetic process often limit
the application of these molecules toward societal problems.1
Improving economies of atoms, chemical steps, and process
operations incurred throughout syntheses has therefore been a
major goal of chemists seeking to develop new chemical
transformations. Ideally, these reactions should enable the
synthesis of target molecules from simple building blocks
without the need for functional group manipulations or
protecting groups.2 Reactions involving catalytic functionali-
zation of carbon–hydrogen (C–H) bonds provide one means
to improve synthetic efficiency, and, as this thematic issue
illustrates, this field has experienced impressive progress.
Catalytic functionalization of C–H bonds presents three
fundamental challenges to chemists: activating an inert C–H
bond, functionalizing the activated organic fragment in a manner
that permits catalyst turnover, and accomplishing both of
these tasks with high chemo-, site/regio-, and stereoselectivity.3
Activation of C–H bonds has been demonstrated on a wide
range of substrates, particularly arenes, using numerous
transition metal complexes, and many methods for catalytic
functionalization have also emerged.4 However, obtaining
suitable selectivity, particularly on functionally complex
compounds, remains highly challenging. The most common
approach has involved using structural and electronic properties
aDepartment of Chemistry, University of Chicago,Searle Chemistry Laboratory, 5735 S. Ellis Ave.,SCL 302, Chicago, IL 60637, USA
bDivision of Chemistry and Chemical Engineering,California Institute of Technology, 1200 E. California Blvd.,MC210-41, Pasadena, CA 91125-4100, USA.E-mail: [email protected]
w Part of a themed issue on C–H functionalization in organic synthesisdedicated to the memory of Keith Fagnou.
Jared C. Lewis
Jared C. Lewis is an AssistantProfessor of Chemistry at theUniversity of Chicago. Hereceived his BS in chemistryfrom the University of Illinois,working with Eric Oldfield,and his PhD from the Univer-sity of California, Berkeley,under the direction of JonathanEllman and Robert Bergman.He carried out postdoctoralresearch in Frances Arnold’slaboratory at the CaliforniaInstitute of Technology.
Pedro S. Coelho
Pedro S. Coelho is a chemistrygraduate student at Caltech,working in the research groupof Professor Frances Arnold.He received his BA andMastersin Chemistry from the Univer-sity of Oxford, where he workedin the laboratory of ProfessorFraser Armstrong.
CRITICAL REVIEW www.rsc.org/csr | Chemical Society Reviews
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of a substrate that render only a single C–H bond of that
substrate (e.g. one proximal to a directing group) reactive
toward a given catalyst. While these methods eliminate the need
for prefunctionalization in some cases, their restricted substrate
scope limits their utility, particularly in complex molecules with
many C–H bonds of similar reactivity.
Researchers have also appended molecular recognition
elements to catalysts capable of activating many substrate C–H
bonds to orient the substrate such that only a single C–H bond
is presented to the catalyst for reaction.5 Unfortunately,
adapting such systems for selective recognition of different
substrates or different C–H bonds on a given substrate
presents significant synthetic challenges. Of course, the synthetic
utility of molecular recognition was long ago discovered in
nature and provides a major component of the amazing rate
enhancements of enzyme-catalyzed reactions, including C–H
functionalization, over their non-catalyzed congeners. Unlike
any synthetic structures explored, however, the activity and
selectivity of enzymes can be systematically optimized for
reaction of a substrate via directed evolution.6
This review is intended to serve as an introduction to the
exciting array of enzyme-catalyzed reactions in which non-acidic
C–H bonds (i.e. not alpha to a ketone or imine) are function-
alized. Only reactions in which C–H bonds are converted to
C–X bonds (X = C, N, O, S, halogen), rather than removed in
desaturation reactions, are included due to space limitations,
and examples are chosen to illustrate mechanistic and reactivity
principles rather than to serve as an exhaustive list. Readers are
directed to many excellent reviews on specific enzyme classes for
more in-depth treatments throughout this article. Also covered
are cases in which this novel reactivity has translated to practical
applications, cases in which protein engineering has facilitated
such applications, and a number of emerging technologies
involving enzymatic C–H bond fuctionalization.
II. Enzymatic C–H bond functionalization
in nature
Heterotrophic organisms generate chemical energy
(ATP, NAD(P)H, FADH2, etc.) from the conversion of
organic molecules into carbon dioxide and water.7 C–H bonds
must necessarily be cleaved during this process, but metabolic
pathways accomplish this task primarily via deprotonation
alpha to carbonyls or imines or via dehydrogenation of
alcohols or amines (Fig. 1).8 While these mechanisms can
completely account for the metabolism of many compounds
(e.g. sugars and lipids), they are powerless against more
inert substrates with aryl or unsubstituted-alkyl C–H bonds.
However, the abundance of such substrates in certain environ-
ments has provided selective pressure for the evolution of
enzymes that can activate a range of C–H bonds with
pKa’s above 50 and bond dissociation energies (BDEs) above
110 kcal mol�1.
While catalytic activation of such bonds remains largely
elusive to synthetic chemists, several different enzymatic
mechanisms have emerged in nature to accomplish this task
at ambient temperature and pressure in aqueous solution.9
Essentially all of these involve the action of cofactors,
frequently metals, positioned within the enzyme active site in
a manner that controls the substrate scope and selectivity of
the reaction. Two broad classes of reactivities are observed
depending on whether functionalization of aromatic or
aliphatic C–H bonds is involved. Aromatic substrates are
unique in that their C–H bonds can be activated via reaction
of their p-electrons.10 For example, attack of aromatic
p-electrons on an electrophile leads to the formation of a
cationic (Wheland) intermediate with significantly enhanced
C–H acidity (Fig. 2A). Deprotonation of this intermediate
leads to re-aromatization and formal substitution of an
aromatic C–H bond by the electrophile to complete the
familiar electrophilic aromatic substitution process. Long
before chemists began exploiting this reactivity for the synthesis
of aniline dyestuffs, enzymes evolved novel means for catalyti-
cally generating electrophiles and enforcing high levels of
regioselectivity to enable efficient functionalization of aromatic
C–H bonds.
Heterocycles and heteroatom-substituted arenes can also be
activated by direct abstraction of their lone-pair electrons to
generate radical intermediates (Fig. 2B).11 These can undergo
a range of addition or rearrangement reactions, ultimately
followed by re-aromatization, to enable functionalization of
C–H bonds. Analogous chemical processes for substitution of
Fig. 1 Typical biological routes to C–H bond cleavage include
(A) deprotonation and (B) dehydrogenation. (C) Mechanism of
alcohol dehydrogenation using NAD(P)H as a hydride acceptor.8
Frances H. Arnold
Frances H. Arnold is theDick and Barbara DickinsonProfessor of ChemicalEngineering, Bioengineeringand Biochemistry at theCalifornia Institute of Techno-logy, where her researchfocuses on evolution of bio-logical systems in the labo-ratory. Her interests includeenzyme evolution and bio-catalysis, particularly forchemicals and fuels fromrenewable resources. She waselected to the US NationalAcademy of Engineering in
2000, the Institute of Medicine in 2004, and the NationalAcademy of Sciences in 2008.
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alkyl C–H bonds are far more energy intensive.10 On the other
hand, the BDEs of alkyl C–H bonds are lower than those of
most arenes, and enzymes have exploited this through the use
of radical intermediates to directly abstract H atoms from
unactivated alkyl compounds (Fig. 2C and D). The resulting
substrate radical can undergo subsequent radical reactions to
functionalize the activated position. Again, unlike synthetic
radical reactions, high selectivity in both inter- and intra-
molecular reactions can be enforced by the enzyme active site.
Using such mechanisms, enzymes are able to catalyze the
formation of a wide range of C–X bonds (X = C, O, N, S,
halogen, etc.). The fate of the reactive intermediates
(i.e. cation, radical cation, or radical) varies greatly depending
on the enzyme and can include subsequent H-abstractions,
radical recombinations, radical or cationic rearrangements,
and many others. These reactions are used in both catabolic
and anabolic processes, the latter of which enable the synthesis
of complex natural products. A variety of examples will be
presented in order to provide a sense for their potential
synthetic utility. The reactivity of these enzymes is largely
dependent on the type of cofactor they employ, so this will
serve as a convenient way to group the enzymes.
III. A survey of relevant cofactors and their host
enzymes
A Flavins and flavoproteins
The flavin nucleotides, flavin mononucleotide (FMN) and
flavin adenine dinucleotide (FAD), are redox-active cofactors
derived from riboflavin via phosphorylation (FMN) and
subsequent adenylation (FAD) (Fig. 3A).12 The chemistry of
these compounds derives from their isoalloxazine heterocycle,
which can undergo two-electron reduction by NAD(P)H
followed by either one-electron-transfer events or two-electron
chemical reactions. Because of this ability, flavins are involved
in a host of biological processes and serve as an important
bridge between polar and radical reactions. Most relevant to
the current discussion, however, is their ability to hydroxylate
C–H bonds of electron-rich (hetero)arenes.13
Hydroxylation of C–H bonds via insertion of a single
oxygen atom derived from dioxygen (monooxygenation) with
concomitant reduction of the second oxygen atom to water
is a key reaction in the metabolism of a huge number of
compounds.14 This requires reduction of dioxygen to a more
reactive state (e.g. superoxide, peroxide, etc.), which in turn
requires overcoming the kinetic barrier imposed by the spin
Fig. 2 Representative mechanisms for enzymatic functionalization of
non-acidic C–H bonds.8,10,11 (A) Electrophilic aromatic substitution.
(B) Electron abstraction followed by radical coupling. (C) H abstrac-
tion followed by radical recombination. (D) H abstraction followed by
radical reaction.
Fig. 3 (A) Structures of riboflavin, FAD, and FMN. (B) Mechanism of nucleophilic and electrophilic oxygenation.13
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mismatch between organic substrates (singlet) and ground
state dioxygen (triplet). Flavoproteins accomplish this by
promoting single electron transfer from reduced flavin
(FADH2) to dioxygen, spin inversion, and radical recombina-
tion to generate a nucleophilic flavin peroxide at C4a of the
isoalloxazine ring (Fig. 3B).15 This C4a peroxide intermediate
can be protonated to generate the corresponding electrophilic
hydroperoxyflavin, a mild oxidant (vide infra) that can serve
formally as a source of ‘‘OH+’’. Within the active site of
flavoproteins, the regioselectivity of this oxidant toward
electron-rich substrates can be controlled. Two broad classes
of flavoproteins, the flavin monooxygenases and the
FAD-dependent halogenases, utilize this oxidant to functionalize
the C–H bonds of electron-rich heterocycles via hydroxylation or
halogenation, respectively.13
Flavin monooxygenases comprise a highly diverse family
of enzymes and are capable of catalyzing many different
oxygenation reactions in addition to C–H bond hydroxyl-
ation, including epoxidation, Baeyer–Villiger oxidation, and
amine oxidation.13 These variations in reactivity can be roughly
correlated with discrete monooxygenase subclasses defined
by sequence and structural differences, including number
of domains and cofactor requirements (FAD/FMN and
NADH/NADPH). Two subclasses defined in this manner
have aromatic hydroxylase activity. These enzymes typically
exhibit a narrow substrate scope and utilize selective substrate
binding to enhance the rate of NAD(P)H-mediated FAD
reduction by up to five orders of magnitude (Fig. 3).12 This
ensures that FADH2 is generated only in the presence of
substrate and minimizes consumption of valuable NAD(P)H
and formation of free reactive oxygen species. Such
control mechanisms are common in enzyme-catalyzed C–H
functionalization reactions and will be highlighted throughout
this review.
One of the most studied examples of these enzymes is
p-hydroxybenzoate 3-hydroxylase (Fig. 4A).16 As previously
mentioned, enzyme-catalyzed reaction of FADH2 with O2
followed by protonation leads to the generation of a flavin
C4a hydroperoxide intermediate and places the reactive
‘‘OH+’’ equivalent proximal to a specific site on the aromatic
substrate. Electrophilic aromatic substitution of the arene
C–H bond with this OH group then provides regioselective
hydroxylation of the substrate. Several enzymes with similar
activity toward other aromatic substrates are also known.13
One recent report describes the isolation and characteri-
zation of an FMN-dependent long-chain alkane hydroxylase,
LadA, most similar to the luciferase subclass of oxygenases
(Fig. 4B).17 Unfortunately, the authors were unable to deter-
mine the mechanism through which active site residues might
enable the use of an FMN-derived oxidant to activate alkyl
C–H bonds.
A variation of the monooxygenase mechanism is used
by FAD-dependent halogenases to effect halogenation of
electron-rich arenes found in a variety of antimicrobial natural
products.18 Several tryptophan halogenases have been charac-
terized, and these are among the most well-studied of the
FAD-dependent halogenases (Fig. 5).19 As their name suggests,
these enzymes catalyze the halogenation of tryptophan, but
they do so at the 5-, 6-, or 7-position of the benzene
ring, rather than the more electron-rich 2-position. In analogy
to the monooxygenase mechanism described above, these
enzymes generate a flavin C4a hydroperoxide intermediate.
However, instead of directly hydroxylating aromatic substrates,
this species reacts with a specifically bound halide anion
(X, X= Br� or Cl�) to generate HOX. In an amazing product
of evolution uncovered by several impressive biochemical
studies on tryptophan 7-halogenases, RebH and PrnA, this
species is proposed to diffuse through the center of the enzyme
where it is intercepted by a conserved active site lysine residue
to generate either a haloamine or hydrogen-bonded HOX
intermediate adjacent to a conserved glutamate residue.20
Specific substrate binding proximal to this intermediate
enables regioselective halogenation of the arene via electro-
philic aromatic substitution, with different binding modes
giving rise to different regioselective outcomes.
B The 50-deoxyadenosyl radical (Ado�) and Ado� enzymes
Flavin-promoted dioxygen activation confers on flavoproteins
the ability to hydroxylate or halogenate C–H bonds of
aromatic compounds. However, excepting the recent example
of alkane hydroxylase activity by LadA, the substrate scope of
these enzymes is generally limited to electron-rich aromatic
compounds by their use of electrophilic aromatic substitution-
based mechanisms. In order to functionalize substrates outside
of this range, a more powerful oxidant is required. Several
solutions to this problem have evolved in nature, and all of
these appear to rely on paramagnetic intermediates to promote
single-electron or H-atom abstraction from substrates followed
by manifold possibilities for subsequent C–H functionalization.8
While many of these enzymes utilize oxygen-derived oxidants,
at least one class of enzymes appears to have evolved
before the oxygenation of earth and are in fact often highly
oxygen-sensitive.21 These utilize a ubiquitous biomolecule,
adenosine, albeit in a unique form, the 50-deoxyadenosyl
radical (Ado�), in order to abstract hydrogen atoms from a
wide range of substrates, including unsubstituted alkanes
(Fig. 6). This cofactor can function either as a stoichiometric
reagent that must be regenerated after each catalytic cycle or
as a catalyst by reversibly abstracting hydrogen from a
substrate. The enzymes containing this cofactor can be
broadly classified based on the means by which the adenosyl
radical is generated, namely via homolytic cleavage of adenosyl-
cobalamin (vitamin B12, AdoCbl)22 or one-electron reduc-
tion of a [4Fe–4S] cluster-complexed S-adenosylmethionine
(AdoMet)23 (Fig. 6). Notably, both of these reactions are
Fig. 4 (A) Primary reactions of p-hydroxybenzoate 3-hydroxylase
(PHBH)16 and (B) a newly-characterized long-chain alkane hydroxylase,
LadA.17
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highly unfavorable, and the free energy for forming Ado� in
each case is approximately 30 kcal mol�1.21a Because the
enzyme readily accomplishes this task, it has been hypothesized
that this barrier is offset by protein-induced weakening of
the Co–C bond (for AdoCbl) or decrease of the reduction
potential of the iron–sulfur cluster (for AdoMet) in response
to substrate binding, again providing a switch to preserve
valuable cofactor in the absence of substrate.
The AdoMet enzymes constitute the largest and most
primitive class of Ado� enzymes, and sequence analysis has
identified approximately 3000 putative members of this family
based on the CxxxCxxC consensus motif.21b These enzymes
are highly oxygen-sensitive due to oxidative decomposition of
their [4Fe–4S] cluster and are most commonly found in anaerobes.
However, their reactivity enables organisms to activate inert
C–H bonds without the need for oxygen-derived oxidants
(vide infra). Reactions promoted by the Ado� intermediate
generally fall into one of the three categories: reversible H
abstraction, irreversible H abstraction, and irreversible H
abstraction from a glycine residue of a cognate protein which
then catalyzes subsequent reversible H abstractions.24
Only a few examples of enzymes in the first category are
known, and all of these catalyze the 1,2-interconversion of H
and a functional group, X (X = OH, NH3+, etc.).21a In
general, these reactions proceed via initial H abstraction by
Ado� to generate AdoH and a substrate radical alpha to X.
Migration of X to the substrate radical then generates a
second substrate radical, which abstracts H from AdoH to
form the rearranged product and regenerate Ado�. Variations
on this scheme are known, and additional cofactors are often
required to promote the observed rearrangement. This class of
enzymes is perhaps best illustrated by lysine-2,3-aminomutase,
which involves imine formation between lysine and the coenzyme
pyridoxyl phosphate (PLP) followed by radical rearrangement
via a three-membered aziridine intermediate (Fig. 7A).25
In most cases, AdoMet is utilized as a reagent (i.e. irreversible
H abstraction is observed).21 Enzymes in this class can
catalyze reactions analogous to atom transfer processes in
synthetic chemistry and enable installation of either sulfur or
methyl groups at unactivated C–H bonds. The best example of
the former involves the installation of the thiophene ring of
biotin (Fig. 7B).26 To accomplish this, the AdoMet enzyme,
biotin synthase (BioB), utilizes Ado� to abstract a hydrogen
Fig. 5 Proposed mechanism of FAD-dependent halogenases (PrnA/RebH) catalyzing halogenation of tryptophan. A hydrogen-bonded HOX
oxidant is depicted.20
Fig. 6 Structures of AdoMet (A) and AdoCbl (B) and mechanism for
generation of Ado� in each case.21
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atom from the C9 methyl group of dethiobiotin. Interestingly,
the resulting substrate radical then reacts with a [2Fe–2S]
cluster to form a C9–S bond with concomitant reduction of
the proximal Fe center. A second Ado�-mediated H abstrac-
tion at C6 generates a substrate radical that reacts with the
remaining Fe–S bond to generate the C6–S bond. Several
additional enzymes are known to utilize similar mechanisms
to install thiols or thioethers at unactivated C–H bonds.21
Methylation of C–H bonds is also catalyzed by AdoMet
enzymes. As before, H abstraction by Ado� generates a
substrate radical, but in these cases, methylcobalamine serves
as a source of methyl radical to install a methyl group at the
activated carbon. One example of this activity, catalyzed
by the enzyme Fom3, is observed in the biosynthesis of the
natural product fosfomycin (Fig. 7C).27 Although the methylene
C–H bond is modestly activated by the ipso hydroxyl group,
direct methylation of this site is nonetheless an impressive feat.
In the final class of AdoMet enzymes covered here, the Ado�
intermediate abstracts a hydrogen atom from a specific glycine
residue of a cognate glycyl radical enzyme.21a The activated
glycyl radical enzymes are capable of catalyzing a number
of C–H functionalization reactions. These typically involve
abstraction of a cysteine S–H bond by the glycyl radical and
subsequent H abstraction from a substrate by the cysteine
radical. A very interesting example of this type of enzyme is
the benzylsuccinate synthase used for anaerobic metabolism of
toluene (Fig. 7D).28 This enzyme catalyzes abstraction of a
benzyl H from toluene and subsequent reaction of the tolyl
radical with fumarate to generate benzyl succinate. Several
additional enzymes are known for anaerobic metabolism of
other substituted aromatics and even aliphatic substrates.21,27
Formally, this reaction is a hydroalkylation of alkenes, a
valuable synthetic transformation.29 Several additional glycyl
radical enzymes are known and catalyze dehydration, deoxy-
genation, and decarboxylation reactions.21
While the AdoMet enzymes are a large and functionally
diverse class of enzymes, the discovery and characterization of
AdoCbl enzymes predated work on the former and has
provided a great deal of mechanistic information regarding
the activity of Ado� intermediates.21a A beautiful and highly
complex molecule with several discrete chemical domains,
AdoCbl, is a relatively rare example of an organotransition
metal complex in nature. Perhaps not surprisingly, the AdoCbl
enzymes are hypothesized to have appeared more recently
than the simpler AdoMet enzymes. The AdoCbl cofactor is
always used catalytically, probably due to the metabolic
expense of its biosynthesis, which makes it a more atom-
economical solution to Ado�-mediated C–H functionalization,
and it is far less oxygen-sensitive, consistent with its evolution
during more oxygen-rich geological past. However, only a
Fig. 7 Examples of Ado� reactions. (A) 1,2-rearrangement catalyzed by lysine-2,3-aminomutase.25 (B) Double C–H abstraction and
sulfur insertion catalyzed by biotin synthase (BioB).26 (C) Radical methylation catalyzed by Fom3 in the biosynthesis of fosfomycin.27 (D)
Hydroalkylation of fumarate catalyzed by benzylsuccinate synthase.28
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handful of these enzymes are known, and all but one are
limited to catalysis of 1,2-H/X interconversions similar to
those discussed for Class I AdoMet enzymes (e.g. Fig. 7A).
The mechanism of these transformations is conceptually
identical following generation of the Ado� intermediate. Specific
examples of AdoCbl enzymes include lysine-5,6-amino mutase,30
glutamate mutase,31 notable for its ability to promote a carbon
skeleton rearrangement, and diol dehydratase,32 which promotes
a rearrangement followed by spontaneous dehydration to
generate a carbonyl compound.
C Mononuclear non-heme metalloenzymes
As previously mentioned, C–H bond hydroxylation is a key
reaction in cellular metabolism. However, the mechanistically-
limited substrate scope of flavin monooxygenases and the
oxygen sensitivity of Ado� enzymes preclude hydroxylation
of many substrates by either of these enzymes. This apparent
gap in reactivity is filled by an expansive list of metalloenzymes
that activate dioxygen to generate oxidants capable of
hydroxylating even the strongest of alkyl C–H bonds and
promoting a number of less common oxidative transformations.9
The active sites of these enzymes range from residue-coordinated
metal ions, most notably iron and copper,33 to enzyme-bound
coordination complexes, such as heme.34
Among the most prevalent of the former are the mono-
nuclear non-heme iron enzymes,35 which utilize as few as two
active site residues to coordinate an Fe(II) ion and thus have as
many as four sites available to coordinate substrates (Fig. 8A).
These enzymes promote a number of oxidative transforma-
tions by tuning the ligand environment around the metal. The
most common coordination sphere consists of three protein
ligands, (His)2–(Asp/Glu), arranged on one face of an octa-
hedron, leaving the three remaining sites of the other face
occupied by water in the resting state Fe(II) form of the
enzyme. Substrate binding triggers the release of water ligands,
opening a vacant coordination site to which dioxygen can be
bound and subsequently reduced to generate an active oxidant.
Typically, two of the four electrons required for reduction of
dioxygen come from a reducing co-substrate (e.g. a-ketoglutarate,tetrahydropterin, ascorbate or NAD(P)H) and the organic
substrate being oxidized provides the other two. In most cases,
the active oxidant is a ferryl complex, but in some special cases
(vide infra) iron–superoxo complexes have been invoked as
intermediates for hydrogen abstraction.36 High-valent iron–oxo
intermediates are capable of promoting a range of C–H
functionalization transformations, namely hydroxylation, halo-
genation, desaturation/cyclization, and electrophilic aromatic
substitution.35
Fig. 8 (A) General mechanism for hydroxylation of C–H bonds on substrates (R–H) by aKG-dependent dioxygenases.35 (B) Hydroxylation
reaction catalyzed by clavaminate synthase (CAS).38 (C) Chlorination reaction catalyzed by halogenase CytC3.39 (D) Oxidative cyclization
reaction catalyzed by an isopenicillin-N synthase (IPNS).40
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The ubiquitous a-ketoglutarate (aKG)-dependent dioxy-
genases are the largest and functionally most diverse subgroup
of mononuclear non-heme iron enzymes.35 These enzymes
are involved in the synthesis of a range of primary and
secondary metabolites in plants, animals, and microorganisms.
aKG-dependent oxidases catalyze the two-electron oxidation
(e.g. hydroxylation) of their primary substrate with the con-
comitant conversion of the aKG co-substrate into succinate
and carbon dioxide to provide the other two electrons required
for reduction of dioxygen. An important feature for the
control of the reactivity of these enzymes is that dioxygen
activation can only occur following binding of the primary
substrate.37 The mechanism is thought to involve binding of
the aKG co-substrate, followed by binding of the primary
substrate, which displaces the last iron(II)-bound water to
enable oxygen binding and activation (Fig. 8A). A well-
characterized member of this family is clavaminate synthase
(CAS) from Streptomyces clavuligerus, which catalyzes three
different steps (hydroxylation, cyclization and desaturation) in
the biosynthetic pathway of clavulanic acid (Fig. 8B).38
Some Fe(II)/aKG-dependent oxygenases also display aliphatic
halogenase activity in the chlorination of terminal methyl
groups of amino acids tethered via a thioester linkage to the
phosphopantetheine arm of peptidyl carrier proteins (PCPs).18
For example, the non-heme iron halogenase CytC3 from soil
Streptomyces, which requires iron, aKG, oxygen and chloride
for its activity, is capable of chlorinating the g-methyl group of
L-2-aminobutyric acid (L-Aba) tethered to the PCP domain
CytC2 (Fig. 8C).39 Exclusive halogenation over hydroxylation
presumably ensues due to the lower reduction potential of the
chlorine radical (Cl� + e� - Cl�, 1.36 V) relative to the
hydroxyl radical (HO� + e� - HO�, 2.02 V).
Isopenicillin-N synthases (IPNS) are evolutionarily related
to aKG-dependent oxidases, and both families have similar
Fe-coordination spheres: (H2O)3Fe(II)(His)2–(Asp).40 IPNS
does not require aKG as a co-substrate, but instead catalyzes
the formation of penicillin N (IPN) through the four-electron
oxidation of the tripeptide, L-d-aminoadipoyl-L-cysteinyl-D-
valine. The 1 : 1 IPN : O2 stoichiometry requires that the
C–H cleavage steps be affected by different intermediates
and, in fact, IPNS was the enzyme for which C–H cleavage
by an iron–superoxo intermediate was first recognized.36 The
first oxidation is promoted by a formally Fe(III)–superoxo
intermediate that cleaves the L-Cys C–H, leading to formation
of the b-lactam ring (Fig. 8D). The second oxidation event is
thought to involve cleavage of the D-Val C–H by an
Fe(IV)–oxo intermediate which subsequently completes the
b-lactam–thiazolidine core of all penicillins.
D Carboxylate-bridged diiron centers and metalloenzymes
Bacterial multicomponent monooxygenases (BMMs) constitute
a second class of enzymes that utilize residue-ligated iron
centers, specifically carboxylate-bridged diiron centers, for
hydroxylation of unactivated C–H bonds.41 These enzymes
are typically found in bacteria living at the aerobic/anaerobic
interface where they consume unactivated hydrocarbons as
their sole source of carbon and energy. Several classes of
evolutionarily-related BMMs have thus far been identified,
including methane monooxygenase (MMO), alkene mono-
oxygenase (AMO), phenol hydroxylase, and alkene/arene
monooxygenase (Fig. 9A).42 BMM systems are composed of
three or four soluble components, namely: a dimeric hydroxylase
protein consisting of two or three subunits in a (abg)2 or (ab)2quaternary structure with the a subunit harboring the carbox-
ylate-bridged diiron site, an NADH reductase, a small effector
protein (necessary for coupling electron consumption with
hydrocarbon oxidation), and in some cases, a Rieske-type
Fig. 9 (A) Representative reactions catalyzed by each of the four classes of bacterial multicomponent monooxygenases (BMMs).41 (B) The
catalytic cycle for the soluble diiron methane monooxygenase.45
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ferredoxin protein (e.g. arene monooxygenases). All components
are required for optimal catalysis and for modulating
regiospecificity.43
The mechanism by which BMMs activate dioxygen and
subsequently catalyze the hydroxylation of C–H bonds has
been studied extensively for the soluble methane mono-
oxygenase from the methanotroph Methylococcus capsulatus.44
Unlike the a-KG dependent enzymes discussed above, in
which iron remained in the +2 oxidation state in both the
resting state and O2-activating forms, the resting state for
methane monooxygenase consists of an oxidized diiron(III)
species (ox) where the positive charge of the two iron atoms
is precisely balanced by four glutamate and two bridging
hydroxide ligands (Fig. 9B). The octahedral coordination
spheres of the two iron atoms are completed by two additional
histidine residues. Reduction of the iron(III) centers in the
resting state to the diiron(II) form (red) is required prior to
dioxygen activation, which is followed by the generation of a
peroxo intermediate, subsequently leading to the fully oxidized
diiron(IV) species, Q.45 Species Q is the active hydroxylating
agent in BMMs and is capable of inserting an oxygen atom
into alkyl C–H bonds, including the 104.5 kcal mol�1 C–H
bond of methane.
While less mechanistic detail is available for the remaining
BMM family members, these enzymes catalyze the hydroxyl-
ation of a number of synthetically relevant compounds. For
instance, the toluene monooxygenases (TMO of the alkene/arene
monooxygenase class) from Pseudomonas mendocina and
Ralstonia pickettii have been well characterized and are known
to display high regiospecificity for the hydroxylation of
toluene to p-cresol (>90% selectivity with only negligible
amounts of benzylic oxidation).43 This preference for aromatic
ring hydroxylation provides an interesting contrast with the
xylene monooxygenases (XMO) for which benzyl oxidations
are predominantly observed.46 The XMO from Pseudomonas
putida is the first enzyme system involved in the degradation of
toluene and xylenes when these hydrocarbons are supplied as a
sole source of carbon and energy.47 For example, toluene is
initially oxidized by XMO to benzylalcohol, which can then be
further oxidized to benzoic acid by the benzylalcohol and
benzylaldehyde dehydrogenases. These three enzyme systems
have been utilized in conjunction to demonstrate the synthetic
potential of converting methyl-substituted aromatics into
carboxylic acids in one pot (Fig. 10).48
E Binuclear copper monooxygenases
Certain bimetallic copper enzymes also possess the ability to
hydroxylate carbon–hydrogen bonds.14 These monooxygenases
can be divided into two classes depending on the extent of
magnetic coupling between the two copper ions. In coupled
binuclear copper monooxygenases (i.e. tyrosinases), the
two copper centers are each ligated by three histidine residues
in close proximity (B3.6 A) to one another. Activation
of molecular oxygen by the copper centers leads to a
m-Z2:Z2-peroxo-bridged dicopper(II) species that exhibits
strong antiferromagnetic coupling.49 In contrast, non-coupled
binuclear copper enzymes (e.g. dopamine b-monooxygenase
(DbM) and peptidylglycine a-hydroxylating monooxygenase
(PHM)) utilize two nonequivalent copper centers, (His)2(Met)CuMand (His)3CuH, distant in space (B11 A in PHM) that exhibit no
observable magnetic interactions.50,51
Tyrosinases catalyze ortho-hydroxylation of monophenols
and subsequent two-electron oxidation of the intermediate
catechols to produce quinones, and the mechanism of these
transformations has been studied extensively.52 In the resting
state of the enzyme, a caddie protein shields the entrance to the
active site to regulate substrate access.53 Activation proceeds
via cleavage of this caddie protein, which exposes the active
site to incoming substrate. Z1-Coordination of the phenolic
hydroxyl group to the bridging m-Z2:Z2-peroxo dicopper(II)
intermediate induces electrophilic attack of the phenol (Fig. 11A).
This generates an o-diphenolate that binds in a bidentate
fashion and is finally oxidized to the quinone with concomi-
tant reduction of the remaining oxygen atom from dioxygen to
water and formation of the deoxy dicopper(I) state.
For the non-coupled dicopper monooxygenases (e.g. DbMand PHM), the binding and activation of dioxygen takes place
at a single copper site (CuM) (Fig. 11B and C). Oxygen
activation is thought to proceed via one-electron reduction
of dioxygen to generate a Z2-superoxo–CuIIM intermediate.
This species can abstract H atoms from aliphatic C–H bonds
to form a CuIIM–hydroperoxo complex and carbon radical.54
Hydroxylation then occurs via recombination of hydroxyl
radical from the hydroperoxo complex and the substrate
radical and leads to the formation of a highly oxidizing
CuIIM–oxyl (O��) species that provides the driving force for
the required long-range intramolecular electron transfer from
the second copper (CuIH). Thus, the role of the CuH site is
simply to provide the additional electron that is necessary to
complete the catalytic cycle.
Recently, the active site of the particulate form of methane
monooxygenase (pMMO) was also shown to consist of a
dicopper center.55 This membrane-associated enzyme is the
most prevalent form of methane monooxygenase and is found
in every methanotroph, while sMMO (vide supra) is only
expressed by some strains of methanotrophs under copper-
limited conditions. The two copper ions in the active site of
pMMO are ligated by three histidine residues and are held in
close proximity (2.5–2.7 A) to one another, but little more is
Fig. 10 Toluene monooxygenase (aromatic oxidation) vs. xylene monooxygenase (benzylic oxidation) and subsequent dehydrogenation for the
synthesis of phenols, alcohols, aldehydes, and acids.48
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known about the mechanism of this enzyme. Computational
studies of methane activation by mixed valent bis-(m-oxo)dicopper (II/III) species56 and reports of direct methane oxida-
tion by mono-(m-oxo) dicopper (II/II) cores in zeolites57 illustrate
the potential of dicopper centers to promote the oxidation of
methane to methanol, but further investigations will be required
to better understand the mechanism of methane activation by
pMMO.58
F Heme and heme enzymes
Heme enzymes are perhaps the most familiar of those capable
of functionalizing C–H bonds.34 The heme cofactor in these
enzymes is comprised of a protoporphyrin IX-bound iron
atom that binds to a (proximal) active site residue leaving a
single (distal) coordination site available for ligand binding
and catalysis. Depending on the nature of the proximal ligand
and active site residues, the heme cofactor can react with
either peroxides or dioxygen to generate complexes capable
of functionalizing C–H bonds.59 Extensive research has been
devoted to studying the formation and reactivity of these
intermediates in different heme enzymes; however, the trans-
formations relevant to the current discussion can at least
formally be described as proceeding via an Fe(IV)–oxo
p-radical cation complex known as compound I.60 This inter-
mediate can react with substrates to functionalize C–H bonds
via several distinct manifolds characteristic of the different
heme enzyme subfamilies. For example, peroxidases catalyze
oxidative coupling reactions via sequential one-electron
abstractions,61 haloperoxidases catalyze halogenation of sp2
C–H bonds via halide oxidation,18 and cytochromes P450
(CYPs) can catalyze hydroxylation of sp2 and sp3 C–H bonds
via H atom abstraction, electrophilic aromatic substitution, or
direct oxo transfer.62
In the case of peroxidases, compound I is readily generated
and can be observed by the reaction of hydrogen peroxide
with the penta-coordinate ferric resting state (Fig. 12A).63
However, even within the peroxidase family, structural
variations lead to pronounced differences in reactivity. For
example, in horseradish peroxidase (HRP), the axial ligand to
the heme is a histidine nitrogen that acts as a hydrogen bond
donor to a nearby aspartate.64 This is thought to impart a
greater imidazolate character on the axial histidine that is
considered important in the enzyme’s ability to activate
hydrogen peroxide and favor sequential one-electron oxida-
tions over two-electron oxo transfer from compound I. This
reactivity, in turn, enables HRP to catalyze oxidative coupling
reactions between electron-rich (hetero)arenes or between a
(hetero)arene and a heteroatom (e.g. Fig. 12B).65
The haloperoxidases are unique among the peroxidases in
having a thiolate-ligated heme. Such a strongly donating axial
ligand is thought to facilitate heterolytic cleavage of the O–O
bond and to lower the reduction potential of the heme
iron.59 The chloroperoxidase (CPO) isolated from mold
Caldariomyces fumago is the most extensively studied halo-
peroxidase; its gene has been sequenced and cloned, functional
heterologous expression has been achieved, and it is even
commercially available as a fine chemical.66 Seminal studies
on this enzyme revealed that it can catalyze the halogenation
of monochlorodimedone, a mimic of the C. fumago natural
product caldariomycin.67 However, CPO is also capable of
promoting oxidative transformations that are characteristic of
other heme enzymes, such as dehydrogenation (i.e. peroxidases),
H2O2 decomposition (i.e. catalases), and oxo transfer reactions
(i.e. CYPs). These reactions, including C–H bond hydroxyla-
tion (vide infra), are catalyzed by CPO in the absence of halide
at neutral pH.
In the presence of halide (X�, X = Cl, Br, I) and low pH
(o3), however, CPO reacts with halide to generate the corres-
ponding hypohalous acid (HOX).18,67 This species can in turn
halogenate a range of electron-rich substrates, including
olefins, aromatics, and b-diketones, via electrophilic substitu-
tion pathways (e.g. Fig. 12C).66 Most data are consistent with
HOX being free to diffuse out of the active site and react with
the substrate in the surrounding medium.66 This is confirmed
by the similar patterns of halogenation observed for various
substrates with CPO versus simply using hypohalous acid free
in solution. Consequently, regio- or stereoselective halogena-
tion is not possible for CPO, and this feature constitutes an
important difference between the haloperoxidases and the
FAD-dependent halogenases discussed above.19
CYPs also possess cysteine as the axial heme ligand
but have a much more hydrophobic active site than the
haloperoxidases.68 And while CYPs can utilize hydrogen
peroxide as an oxidant via a so-called ‘‘peroxide shunt’’ pathway,
they are distinguished from haloperoxidases by their ability to
Fig. 11 (A) The catalytic cycle for tyrosinase.52 Reactions catalyzed
by (B) dopamine b-monooxygenase (DbM) and (C) peptidylglycine
a-hydroxylating monooxygenase (PHM) (AscH2 = ascorbic acid,
Pep = D-Tyr-Val).50,51Dow
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access compound I via dioxygen activation.69 The first step in
this process involves displacement of a water ligand from the
low-spin Fe(III) heme resting state by substrate to generate a
predominately high-spin substrate-bound complex (Fig. 12A).
In many systems, this spin shift is required to increase the
heme reduction potential so that it can be more easily reduced
to the corresponding Fe(II) complex, which minimizes the
expenditure of metabolically valuable reduced cofactors in
the absence of substrate. Oxygen binding to the Fe(II) inter-
mediate leads to the formation of an Fe(III)–oxy complex
that undergoes one-electron reduction, two sequential distal
oxygen protonation events, and elimination of water to
generate compound I. This species can then react with sp3
C–H bonds of the bound substrate via formal H atom
abstraction to generate an Fe(IV)–hydroxyl complex and a
substrate radical followed by radical rebound to provide
the hydroxylated product and the Fe(III) heme complex
(e.g. Fig. 12D). Compound I has been proposed to react with
substrates via a variety of pathways, including electrophilic
substitution and concerted oxo transfer,62 and similar reactivity
is observed for this intermediate in CPO.66
The more than 11 500 distinct CYPs identified thus far are
capable of hydroxylating a wide range of organic substrates
and play important roles in both biosynthetic and degradative
pathways.70 Despite their broad substrate and reaction scope
and low amino acid sequence identities, the CYP super-family
is united in having a conserved fold/structural topo-
logy. Evolution has demonstrated the versatility of this
platform by utilizing CYPs to promote a range of additional
transformations including epoxidation, oxidative deformyl-
ation, dehydrogenation, rearrangements, Baeyer–Villiger oxy-
genation, and oxidative decarboxylation.71
IV. Enzymatic C–H functionalization as a
synthetic tool
A Synthetic utility of enzymes
The enzymes highlighted in the previous section functionalize
a range of C–H bond types via a variety of chemical transfor-
mations without directing or protecting groups. While the
development of such catalysts constitutes a highly sought-after
goal of synthetic chemistry,3 practical application of enzymes
requires overcoming a variety of challenges regarding enzyme
expression/purification, cofactor supply, organic solvent
and oxygen tolerance, stability, and substrate scope.72 None-
theless, enzymes, including many capable of functionalizing
C–H bonds, are increasingly utilized on an industrial scale for
the production of fine chemicals.73
Here, a number of examples from the literature in which
enzymes are used to functionalize C–H bonds of non-natural
substrates are presented. Importantly, these examples high-
light the fact that an enzyme often exhibits activity on a range
of substrates. Although this activity may not be sufficient for
practical applications, it provides an important first step
toward the development of such catalysts.74 It is also worth
noting that many of the enzyme classes discussed above have
not yet been successfully employed as biocatalysts due to one
Fig. 12 (A) Structure of a heme cofactor and general schemes for its conversion to compound I in peroxidases (HRP and CPO) and cytochromes
P450 (CYPs).60 (B) Oxidative coupling catalyzed by HRP.65 (C) Halogenation catalyzed by CPO.66 (D) Hydroxylation catalyzed by CYP 102A1.
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or more of the aforementioned technical challenges.14 The
successes outlined herein will, we hope, provide impetus
for further research into overcoming these difficulties so that
additional enzyme-catalyzed reactions will be available to
chemists.
1 FAD-dependent hydroxylases and halogenases. Flavin
monooxygenases have been extensively investigated as bio-
catalysts due to the importance of aromatic hydroxylation and
the difficulties associated with chemical methods for this
reaction.75 Because most of these enzymes are bacterial in
origin, they are readily expressed in convenient recombinant
hosts such as E. coli, which greatly facilitates their investi-
gation. Several FAD-dependent hydroxylases have been utilized
for hydroxylation of electron-rich aromatic compounds
structurally similar to their native substrates. In one example,
purified 2-hydroxybiphenyl 3-monooxygenase (HbpA) from
Pseudomonas azelaica HBP1 was used to selectively hydroxy-
late a range of 2-alkyl-, aryl-, or halo-substituted phenols to
provide the corresponding 3-substituted catechols (Fig. 13A).76
This enzyme was subsequently expressed in E. coli and used as a
whole-cell biocatalyst for the production of kilogram quantities
of catechols.
In a second example, the substrate scope of p-hydroxy-
benzoate 3-hydroxylase from Rhodococcus rhodnii 135 and
opacus 557 was explored (Fig. 13B).77 This work demonstrated
that a variety of 2- and 3-substituted 4-hydroxybenzoate
substrates could be hydroxylated and that the regioselectivity
of hydroxylation varied greatly between the two enzymes
investigated. Such variation clearly indicates the potential
for modification of selectivity for C–H functionalization in
enzymes (vide infra). While many additional examples of arene
hydroxylation by FAD-dependent enzymes are known,75 these
two examples represent the types of reactions and substrate
scope that have been explored.
On the other hand, relatively little work has been published
regarding synthetic applications of FAD-dependent halogenases.
These enzymes were only detected in the late 1990’s, and the
first example in which the activity of a member of this family
was reconstituted in vitro appeared in 2000.19 Since that time,
several such enzymes have been characterized, and extensive
mechanistic investigation, particularly in the case of the
tryptophan halogenases, has clarified their mode of action
as described in the previous section.20 Building on these
advances, van Pee and co-workers have explored the substrate
scope of one tryptophan-7-halogenase, PrnA (Fig. 14).78 A
variety of substituted indoles were accepted by the enzyme,
although halogenation invariably occurred at the electroni-
cally most activated indole C2–H bond for all but tryptophan.
2 Ado� dependent dehydratases. While Ado� enzymes
catalyze an enticing array of reactions, their oxygen sensitivity
and cofactor (Cbl or Met) requirements certainly pose
challenges to their practical application.21 A notable exception
to this trend involves the use of recombinantly expressed
dehydratases to convert glycerol to 3-hydroxypropionaldehyde.79
This reaction has been explored by several researchers and
recently utilized in a commercial process for production of
1,3-propoanediol, a valuable commodity chemical (vide infra).
Cameron and co-workers provided a thorough description of
a similar process employing an AdoCbl-dependent glycerol
dehydratase and an oxidoreductase from Klebsiella pneumoniae.80
This sequence involves dehydratase-catalyzed conversion of
glycerol to 1,1,3-propanetriol via an H-abstraction/rearrangement
mechanism similar to those shown in Fig. 7.32 Spontaneous
dehydration and subsequent reduction of the intermediate
Fig. 13 Synthesis of substituted phenols using (A) 2-hydroxybiphenyl
3-monooxygenase (HbpA)76 and (B) p-hydroxybenzoate 3-hydroxy-
lase (PHBH).77Fig. 14 Substrate scope and regioselectivity of indole halogenation
using the halogenase PrnA.78
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3-hydroxypropionaldehyde generates 1,3-propanediol. In addi-
tion to its commercial utility, this example provides valuable
precedent for the development of other processes employing Ado�
enzymes.
3 Non-heme iron enzymes. Of the non-heme iron enzymes
capable of functionalizing C–H bonds, BMM, and in particular
the toluene and xylene monooxygenases (TMO and XMO),
have enjoyed the most attention in biocatalysis applications.43
As previously described, the native activity of these enzymes
involves aromatic and benzylic hydroxylation, respectively.
Examination of the substrate scope of these enzymes has
revealed that both possess activity on a broad range of aromatic
and aliphatic compounds.81
The reactivity of TMO has been exploited by a number of
researchers for the hydroxylation of various toluene derivatives.
For example, recombinant toluene-4-monooxygenase (T4MO)
was used to hydroxylate m-, o-, and p-xylene, benzene, chloro-
benzene, anisole, and nitrobenzene with at least 90% selectivity
(Fig. 16A).82 Only trace amounts of benzylic hydroxylation
were observed, and even the slowest reactions proceeded at a
rate within a factor of 3 of the native reaction. This activity
was exploited in later syntheses of 4-fluorocatechol83 and
hydroxytyrosol.81 XMO also accepts a range of toluene
derivatives, and whole-cell biocatalysts expressing this enzyme
(P. putida mt-2) have been used for the benzylic oxidation of
these compounds (Fig. 16B).84
These enzymes have also been used for commercial processes.
For example, Lonza used P. putida ATCC 33015, which
harbors an XMO and additional dehydrogenases for benzylic
hydroxylation and subsequent alcohol and aldehyde dehydro-
genation to convert a range of methyl-substituted heterocycles
to the corresponding carboxylic acids (Fig. 16C).48 The bio-
conversions provided high yields (40–90%) for a range of
heterocycles in large-scale fermentations (up to 24 g L�1, 95%
conversion, 1000 L scale). While selective oxidation of C–H
bonds is a challenging task in its own right, this example is
even more impressive in that it uses heterocycles, which are
frequently challenging substrates for synthetic metal-catalyzed
reactions.
4 Heme enzymes. By far the most work on enzymatic
functionalization of C–H bonds has focused on heme-containing
enzymes and on cytochromes P450 in particular. Several
excellent reviews have appeared specifically detailing the use
of P450 enzymes as biocatalysts, and readers are directed to
those sources for ample discussion of this subject.72,73 It is
worth emphasizing that these truly remarkable enzymes can
exhibit broad substrate scope while maintaining high reaction
rates and selectivities. While aliphatic C–H hydroxylation
certainly dominates the reactivity of these enzymes, they are
known to catalyze myriad additional transformations, including
C–C bond formation via oxidative coupling.71 There will
undoubtedly be significant practical advances to improve the
utility of these reactions, but several examples already highlight
their synthetic potential.
A number of early studies focused on the use of whole-cell
catalysts harboring various P450 enzymes for the hydroxyl-
ation of steroids. Several of these are used commercially,
including for the conversion of 11-deoxycortisol to hydro-
cortisone using P450lun in Curvularia lunata (Fig. 17A).85 This
example clearly shows how remote, unactivated C–H bonds
can be efficiently hydroxylated even in the presence of an olefin
and acidic, allylic, and tertiary C–H bonds. Bristol Myers
Squibb utilized CYP105A3 in Streptomyces sp. Y-110
to convert compactin to pravastatin (Fig. 17B).86 And finally,
Fig. 15 Production of 1,3-propanediol by E. coli expressing an
AdoCbl-dependent dehydratase and an oxidoreductase.80
Fig. 16 (A) Synthesis of phenol derivatives using recombinant
toluene-4-monooxygenase (T4MO).82 (B) Synthesis of substituted
benzyl alcohols using cells expressing a xylene monooxygenase
(XMO) with reaction rates shown.84 (C) Lonza synthesis of (hetero)-
arylcarboxylic acids using XMO and additional dehydrogenases.48
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a rare example in which a P450 was used for the synthesis
of a bulk chemical is the conversion of n-dodecane to
a,o-dodecanedioic acid by Cognis Inc. (Fig. 17C).87
Other heme-containing enzymes, including chloroperoxidase
(CPO) and horseradish peroxidase (HRP), have also been
explored extensively for a variety of C–H functionalization
reactions.66,61 CPO has been utilized for hydroxylation88 and
halogenation89 (Fig. 18A). On the other hand, HRP can
catalyze oxidative coupling of electron-rich arenes, a reaction
similar to the impressive arylation chemistry developed by
Stuart and Fagnou.90 In one synthetic example, arbutin and
gentisate are regioselectively coupled to generate a tricyclic
compound (Fig. 18B).91 This example highlights the power of
enzymes to provide selectivity to otherwise unselective radical
coupling processes and in this case enable selective C–C bond
formation between two C–H bonds!
B Optimization of enzymes via directed evolution
Enzymes are genetically encoded polymers of amino
acids assembled by the ribosomes of all living organisms. As
discussed above, the ubiquity of this biosynthetic machinery
often allows the gene that encodes an enzyme to be introduced
into heterologous microbial hosts that facilitate its production
at high levels and biocatalytic applications of that enzyme.
Genes can also be readily amplified from genomic DNA
using standard polymerase chain reaction techniques and even
synthesized de novo, which provides access to any enzyme for
which sequence information has been obtained (although
expression of functional enzymes is not always possible).
Furthermore, it is possible to alter the amino acid composition
of recombinant enzymes in a process conceptually analogous
to the synthesis of various small molecule catalysts. Whereas a
particular modification to the latter may require tedious
alteration of synthetic routes, however, such changes, mutations,
are trivial to introduce into enzymes, and a microbe will do the
‘‘synthesis’’ for you (and a little sugar).
The three-dimensional (tertiary) structure of enzymes enables
substrate binding, ideal placement of all manner of functional
groups to promote reaction of these compounds, and dynamic
fluctuations whose implications for catalysis have only recently
begun to be unraveled.92 Rational manipulation of these
catalysts is therefore a highly challenging and vigorously
pursued goal. On the other hand, the genetic encoding of
these catalysts provides a straightforward means to generate
libraries of structurally diverse mutants from a selected parent.
With a means to assay these mutants for a desired property,
improved variants can be identified from a population.6
Mutations can be made randomly or they can be focused
toward particular parts of the protein, based on some hypothesis
regarding how those residues might modify enzyme function.93
Individual library members (i.e. mutant enzymes) can be
arrayed in microtiter plates using a variety of automated
systems. The biggest challenge is usually assaying these library
members to identify those with improved properties relative to
the parent. Most commonly, this is accomplished using a
screen, in which library members are individually assayed.
This is often a highly challenging task and frequently limits the
Fig. 17 (A) Hydroxylation of 11-deoxycortisol using cytochrome
P450lun in C. lunata.85 (B) Hydroxylation of compactin using cyto-
chrome 105A3 in Streptomyces sp. Y-110.86 (C) Six sequential oxida-
tions on n-dodecane catalyzed by cytochrome 52A1 in Candida
tropicalis.87
Fig. 18 (A) Various hydroxylation and halogenation reactions
catalyzed by chloroperoxidase (CPO).88,89 (B) Oxidative coupling
between arbutin and gentisate catalyzed by HRP followed by sponta-
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library size, although libraries need not be exhaustively
screened in order to identify improved variants, and improve-
ments in ultra high pressure liquid chromatography and fast
gradient gas chromatography instrumentation are facilitating
this process.74
Most importantly, the genes encoding variants with improved
properties can be subjected to subsequent rounds of mutagenesis
and screening in order to systematically improve their properties.
This process of ‘directed evolution’ has been successful in
improving the organic solvent tolerance and stability of
enzymes.6 It has also been used to alter or broaden substrate
scope and to improve activity or selectivity on a particular
substrate. Thus families of enzymes can be engineered to
provide starting points for rapid optimization of activity
toward a given target.94 This is a major difference between
enzymes and small molecule catalysts, but a potentially
advantageous one given that a general small molecule catalyst
is often not optimal for a particular application or particularly
amenable to optimization for that application.
Generalizability is an especially valuable trait of catalysts
for C–H functionalization, since such catalysts could poten-
tially be tuned to recognize any desired substrate and provide
high selectivity for the functionalization of any C–H bond
on that substrate rather than relying on substrate-specific
properties for activity or selectivity. An instructive example
of this process from our own laboratory involves the directed
evolution of cytochrome P450BM3 (CYP 102A1), an enzyme
that naturally catalyzes sub-terminal hydroxylation of long-
chain fatty acids, to hydroxylate propane (Fig. 19A).95 A
combination of random mutagenesis of just the P450BM3 heme
domain, screening, and recombination of beneficial mutations
led to mutants with improved activity on medium-chain
alkanes. However, the stability of these enzymes and their
coupling between NADPH consumption and product forma-
tion decreased. These issues were addressed by evolving the
heme, FMN, and FAD domains individually in the context of
the holoenzyme and then fusing them in a final step to
generate P450PMO. P450PMO provided more than 45 000
turnovers on propane and produced 2- and 1-propanol in a
9 : 1 ratio. This enzyme displayed activity on propane com-
parable to that of P450BM3 on its natural substrates and 98.2%
coupling of NADPH consumption to product hydroxylation.
This example shows that enzymes can be evolved to generate
near-native activity on completely foreign substrates.
Investigation of the substrate profiles for variants along the
evolutionary lineage of P450PMO revealed that the specificity
of P450BM3 for hydroxylation of long-chain alkanes was
relaxed in intermediate variants (Fig. 19B).96 The broad
substrate scope of these variants suggested that hydroxylation
of substrates not included in the original activity screens might
be possible. This proved to be the case, and a panel of enzymes
along the P450PMO lineage has enabled rapid hydroxylation
and derivatization of common organic scaffolds that would be
difficult to accomplish using standard chemical means.97
For example, enantioselective hydroxylation of phenyl acetic
esters (Fig. 20A),98 regioselective hydroxylation of various
pharmaceuticals (Fig. 20B),99 chemoenzymatic fluorination
(Fig. 20C),100 and chemoenzymatic elaboration of mono-
saccharides (Fig. 20D)101 have all been accomplished. Specific
transformations were easily improved by further directed
evolution of variants possessing broad substrate scope. These
examples demonstrate the remarkable ability of cytochrome
P450 to adapt to new challenges.
Directed evolution has also been used to improve the
activity and alter the regioselectivity of non-heme iron mono-
oxygenases. In one example, Wood and co-workers utilized
Fig. 19 (A) Total turnovers for variants along the P450PMO lineage on propane and ethane.95 (B) Activity of variants along the P450PMO lineage
on Cn (n = 1–10) alkanes.96
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saturation mutagenesis of sites identified via random muta-
genesis and various biochemical studies to identify variants of
toluene p-monooxygenase (TpMO) from R. pickettii PKO1
capable of selectively hydroxylating toluene at the ortho, meta,
and para positions (Fig. 21).102 These impressive changes in
selectivity were achieved by varying only three active site
residues and highlight the ease with which the selectivity of
enzymes for activation of a particular C–H bond may be tuned.
FAD-dependent monooxygenases have also been improved
by directed evolution. As previously discussed, 2-hydroxy-
biphenyl 3-monooxygenase (HbpA) catalyzes the ortho
hydroxylation of various 2-substituted phenols to provide the
corresponding catechols (Fig. 13A).103 Meyer and co-workers
utilized 1–2 rounds of random mutagenesis and screening to
identify HbpA variants with improved activity on several
substrates, including 2-sec-butylphenol, 2-methoxyphenol, and
2-tert-butylphenol. The authors also observed a significant
increase in NADH coupling in one case. Such improvements
could enable efficient enzymatic hydroxylation of substrates
that differ significantly from 2-hydroxybiphenyl.
V. Future prospects
As discussed above, enzymes enable a range of C–H function-
alization reactions that are not possible using small molecule
catalysts. While a wide array of such enzymes are known, only
a fraction of these have been utilized for biocatalysis, and even
fewer have been adopted for commercial applications or have
undergone engineering to alter their properties. Additional
research on enzyme, process, and metabolic engineering is
therefore required in order to understand, optimize, and
further exploit this reactivity.72 The development and optimi-
zation of the enzymes themselves also remains a central
challenge. Identifying novel enzymes, developing robust
expression protocols, and improving these enzymes for specific
applications are all required. Furthermore, evaluating the
process compatibility of these catalysts must be a part of
these efforts as early as possible.74 Thus, issues regarding
enzyme solubility, stability, organic solvent tolerance, cofactor
regeneration, and substrate supply/product removal must all
be addressed.
As such issues are resolved, many potential applications will
emerge. Among these are the potential to utilize these catalysts
in concert with other enzymes, either in vitro or in vivo, for the
synthesis of complex molecules (Fig. 22A).104 Enzymatic
cascades constructed in vitro enable precise control of all
reaction parameters (e.g. pH, concentration, solvents, etc.)
Fig. 20 (A) Hydroxylation of phenylacetic esters.98 (B) Regio-
selectivity of hydroxylation reactions catalyzed by BM3 chimeras.99 (C)
Tandem P450BM3-catalyzed hydroxylation/DAST deoxyfluorination.100
(D) Heteroatom demethylation for regioselective deprotection of
monosaccharides.101
Fig. 21 Regioselective hydroxylation of toluene using mutants of
toluene p-monooxygenase (TpMO).102
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while avoiding toxicity and purification issues associated with
the use of enzymes in cells. However, this approach also
requires expression and isolation of large quantities of the
desired enzymes and a means to continually supply appropriate
cofactors (e.g. NAD(P)H, FADH2, etc.).
Because of these problems, construction of enzymatic cascades
in vivo via metabolic engineering is currently more common,
and several instances in which C–H bonds on biosynthetic
intermediates are functionalized have been reported.105 For
example, researchers at the CNRS and Aventis Pharma-
ceuticals generated an artificial metabolic pathway comprised
of 13 engineered genes, including those encoding three cyto-
chromes P450 for C–H hydroxylation, for the total biosynthesis
of hydrocortisone from glucose or ethanol (Fig. 22B).106
Keasling and coworkers reported heterologous expression of
a plant cytochrome P450 in yeast to catalyze the allylic
hydroxylation and subsequent oxidation of amorphadiene to
produce artemisinic acid, an intermediate in the production
of the antimalarial compound artemisinin (Fig. 22C).107
Finally, an AdoCbl-dependent dehydratase-catalyzed reaction
similar to that shown in Fig. 15 was utilized in an engineered
pathway for production of 1,3-propanediol (Fig. 22D)
from glucose in a joint venture between DuPont and Tate &
Lyle BioProducts.108
In cases where wild-type enzymes are not available or
otherwise are incompatible with a given process, engineered
enzymes may be of great utility. Furthermore, such enzymes
could enable the biosynthesis of natural product derivatives
with novel biological activity or other valuable properties
without the need for conventional total synthesis. While
protein engineering can be used to optimize enzymes and
metabolic engineering allows enzymes from different organisms
to be combined in robust hosts for the production of desired
chemicals, chemists still enjoy a much broader palette of
reactions than that found in nature. Many of the enzymes
discussed herein utilize the unique reactivity of metal ions or
complexes to effect the functionalization of C–H bonds, but
many additional C–H functionalization reactions could be
enabled through the use of non-biogenic metals, such as
rhodium or ruthenium. Efforts to create artificial metallo-
enzymes by incorporating such catalysts into protein scaffolds
have been ongoing for many years.109 Such constructs could
provide a means to confer selectivity to otherwise unselective
C–H functionalization catalysts. Furthermore, appropriate
design of artificial metalloenzymes could allow the optimi-
zation of these catalysts using the well-established directed
evolution techniques outlined above.110 This would greatly
facilitate chemists’ ability to tune the selectivity of a catalyst
Fig. 22 (A) General scheme for in vitro or in vivo biocatalytic cascades.104 Engineered metabolic pathways for production of (B) hydro-
cortisone,106 (C) artemisinic acid,107 and (D) 1,3-propanediol.108
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for functionalization of any desired C–H bond on a given
substrate.
VI. Conclusion
The enzymes outlined herein have the potential to significantly
improve the efficiency of chemical synthesis by enabling C–H
functionalization reactions not possible with small molecule
catalysts. Despite their diverse structures and modes of action,
many of these enzymes share common traits, including (1)
substrate-dependent generation of (2) reactive electrophilic or
radical intermediates with (3) selectivity imposed by molecular
recognition of the substrate. Perhaps most importantly, these
catalysts, like all enzymes, can be evolved to better suit
particular applications and can be used to impart microbes
with the ability to produce novel compounds. Thus, many of
the features that make these catalysts so powerful are now
being utilized to develop artificial systems to further expand
the range of transformations possible. Continued effort on all
of these fronts will lead to improved processes employing
enzymes for the functionalization of C–H bonds and for
simplifying the synthesis of complex molecules.
Acknowledgements
Prof. Keith Fagnou’s work in the field of C–H bond function-
alization and on direct arylation in particular provided many
exciting insights into mechanistic aspects of C–H activa-
tion chemistry and led to the development of a number of
conceptually novel transformations. His constant advances
inspired me to work faster and think harder. He will be
missed—JCL.
JCL is supported by a U.S. National Institutes of Health
Pathways to Independence Award (1K99GM087551-01A1).
This work was supported by the U.S. National Institutes of
Health (2R01GM068664-05A1) and by the U.S. Department of
Energy, Office of Basic Science, grant DE-FG02-06ER15762.
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